This application claims priority from U.S. Provisional Application Ser. No. 60/213,987, filed on Jun. 25, 2000, entitled Optically Active Substrates, which is incorporated by reference.
The present invention relates to detecting and analyzing biological materials by optical scanning, and particularly relates to optical scanning or imaging of biological materials located on optically active substrates.
Microarray technology enables studying complex biochemical reactions and systems at once instead of studying them individually. The technology provides a massively parallel form of analysis that increases data collection per unit time, decreases the overall time required for analysis, uses smaller sample volumes and reagent volumes and sometimes reduces disposable consumption. Although the initial cost may be high, overall the technology represents considerable savings in the time and costs of associated labor. Microarray technology became a fundamental tool for genomic research. The technology can also be utilized for routine analysis used in clinical diagnostics or for industrial analytical purposes.
In general, microarrays can be created by optical (or other radiation) directed synthesis, or by microfluidic delivery of nucleic acids onto different substrates. The first technique uses photolithography or other submicron technologies to define positions at which single specific nucleotides are added to growing single-stranded nucleic acid chains. Series of precisely defined nucleotide additions and light directed chemical linking steps are used to synthesize high-density arrays of defined oligonucleotides on a solid substrate. A microarray of probe sequences may be fabricated by using techniques described in U.S. Pat. No. 5,143,854 or PCT Application published as WO 92/10092, or U.S. Pat. Nos. 5,384,261; 5,405,783; 5,412,087; 5,424,186; 5,445,934; 5,744,308 all of which are incorporated by reference. Alternatively, microarrays can be fabricated by other techniques as described in PCT Application PCT/US99/18438 published as WO 00/09757, which is incorporated by reference.
According to a second technique, microarrays are created by microfluidic delivery, as described in PCT Application PCT/US99/00730, published as WO 99/36760, which is incorporated by reference. These microarrays can contain a wide range of biological materials including, plant, animal, human, fungal and bacteria cells; viruses, peptides, antibodies, receptors, and other proteins; cDNA clones, DNA probes, oligonucleotides, polymerase chain reactions (PCR) products, and chemicals. These biological materials are delivered in form of an array of spots to various microarray substrates including chemically treated glass microscope slides, coverslips, plastics, membranes, or gels. The number of deposited spots is in the range of 100 to 50,000 per microarray, and the diameter of an individual spot is in the range of 50 μm to 1000 μm, and preferably 100 μm to 250 μm. The volume of each deposited spot is in the range of 10 pL to 10 nL, and preferably 50 pL to 500 pL.
There are several types of microarray scanning and imaging systems for viewing the entire microarray. Myles et al. described different optical scanning and imaging systems in Microarray Biochip Technology, pp. 53–64, edited by M. Schena, published by BioTechniques Books, Natick, Mass. These scanning systems utilize XYZ stage scanning with a stationary microscope objective, pre-objective scanning, or flying objective scanning with a translation stage. Some fluorescence microscopes use a CCD array imager as a detector. Numerous examples are described in the Handbook of Biological Confocal Microscopy edited by James Pawley, Plenum Press, 1989 and 1995, or by Sampas in U.S. Pat. No. 5,900,949.
Many of the above-mentioned instruments are epifluorescent pseudo-confocal laser scanning microscopes. They are pseudo-confocal systems because they have two optical paths. The first path is the excitation path (also called laser path) that defines the pixel size, typically between 3 and 10 μm. This path has a relatively low numerical aperture, around 0.1, and consequently a greater depth of field. The second path is the emission path (also called detection path), designed to maximize energy collection and therefore has the highest possible numerical aperture; however, this provides a small depth of field. A true confocal microscope uses the same numerical aperture for both lenses and a relatively small depth of field, which is used to create “optical sections” of three-dimensional structures. The confocal microscopy is used to reject out of focus background signal.
The depth of field expresses the axial tolerance in locating the sample in order to obtain a valid measurement of a picture element (i.e., a pixel) needed to construct an image. The depth of field is also determined by the necessity to accommodate some irregularities of the large size of the slide or sample inspected. The depth of field (DOF) of a microscope is a function of the energy collection capability of the objective lens as defined by its numerical aperture (NA) and the light wavelength (λ), wherein DOF≈λ/(NA)2. The desired resolution of an image depends on the spot size (d) of the optical system expressed as a function of the numerical aperture (NA) of a perfect objective and the light wavelength (λ), wherein d≈λ/NA.
The energy an optical system gathers in order to have a meaningful signal from the detector is expressed at a first approximation by the second power of the numerical aperture of the objective lens. The numerical aperture is a function of the lens geometry and the index of refraction (n) of the medium, i.e., NA=n sin θ, where θ is the angle formed by the radius of the lens and its focal distance, and n=1 in air. For example, for an objective with NA=0.7 used with visible light, DOF is about 2 micron; however, an objective with NA=0.2 collects less than about 1/10 of light as the objective having NA=0.7, but it has DOF=37 microns.
Practical considerations for microarray scanning require a large depth of field to accommodate a lens, a scanning stage, substrate flatness and tolerances, and other imperfections. Consequently, there has to be a compromise between the amount of light collected (i.e., the size of NA) and the required depth of field. That is, microarray scanning and imaging instruments require several performance trade-offs.
Fluorescence microscopy is a relatively inefficient process, wherein the light source-to-detector efficiency is estimated in parts per trillions. There is usually a very low efficiency of the fluorescence conversion. Furthermore, among other limitations, the scanning systems cannot increase the intensity of the illumination by the laser source, because the fluorescent sample would be destroyed; this is known as photo-bleaching. Also, before photo-bleaching takes place, most fluorophores behave in a non-linear and possibly unpredictable manner. Additionally, numerous non-optical constrains come into play such as acceptable scan duration, detector performance, and electronic and image manipulation processes.
Therefore, there is a need for optical scanning or imaging systems with high source-to-detector efficiency and for sample substrates and packaging that efficiently utilize light for optical scanning and imaging.